1. Field of the Invention
Vaccination with live attenuated strains is extensively and successfully used in the prevention of various viral diseases of man, such as polio, smallpox, and measles. By contrast, live vaccines are used against only a few bacterial diseases of man or domestic animals: BCG vaccine for prevention of tuberculosis, strain 19 vaccine against bovine brucellosis and Sterne's spore vaccine against anthrax in cattle. Yet in many investigations of experimental Salmonella infections live vaccines have shown advantages over killed vaccines: (i) They frequently prevent, rather than merely postpone, multiplication of Salmonella in liver and spleen, which multiplication may lead to death; (ii) they provide good protection against challenge by oral route, in situations where killed vaccines, given by injection or orally, are relatively ineffective; (iii) in some instances injections of live vaccine confer ability to rapidly eliminate challenge bacteria from liver, spleen, etc., presumably through cell-mediated immunity, in contrast to killed vaccines which evoke mainly humoral immunity, without much ability to eliminate virulent bacteria. The use of live Salmonella vaccines, however, is hampered by a number of factors. Some strains considered for use as live vaccines retain an unacceptable degree of virulence, by reversion or otherwise. Some live vaccines display short persistence of immunity attributed to early disappearance of the vaccine strain from host tissues and, in some instances, incomplete immunity so that some vaccinated animals die after a large challenge inoculum of a virulent strain.
The non-virulent strains used as vaccines have been obtained in various ways. The BCG strain was derived by empirical procedures during prolonged in vitro cultivation, and probably owes its non-virulence to multiple unidentified mutations. Sterne's Bacillus anthracis spore vaccine is a strain which has lost the ability to syntheisze the polypeptide capsule, important as a determinant of virulence but not as a "protective" antigen. Some experimenters have used as live vaccine merely a sublethal dose of a "wild" strain of relatively low virulence in the sense that the LD50 was a large number of bacteria--a situation in which there is evident risk of severe of fatal infection developing in some vaccinated subjects and of transmission to other hosts.
Recently, bacterial strains have been developed for use as live vaccines which are streptomycin-dependent mutants of strains of several pathogenic species. Shigella flexneri and Shigella sonnei streptomycin-dependent mutants have been extensively used as live vaccines given by mouth for protection and have been to be both safe and efficient. In experimental Salmonella infections, however, streptomycin-dependent mutants seem to have been only moderately satisfactory. In general "rough" mutants in Gram-negative bacterial species, i.e., mutants unable to manufacture normal lipopolysaccharide are non-virulent but have proven unsatisfactory as live vaccines because of failure to cause protection. Two exceptions may be noted. (i) In Salmonella mutation of gene ga1E prevents normal lipopolysaccharide synthesis unless the bacteria are provided with preformed galactose. A ga1E mutant of S. typhimurium was virtually non-virulent in small laboratory animals but evoked good immunity. As anti-O antibodies were produced the ga1E bacteria must have obtained sufficient galactose within the host tissue for them to make at least some O-specific lipopolysaccharide. Recently a ga1E mutant of S. typhi, given by feeding to human volunteers, proved non-virulent and conferred reasonable protection against later oral challenge with a virulent strain of the same species. Furthermore, first reports of a field trial of this strain, given by oral route to school children in Alexandria, Egypt, indicate that it gave very good protection against the risk of contracting typhoid fever, which has a high incidence in such children. The non-virulence of ga1E strains seems to be conditional on the presence of normal host cellular defense mechanisms, since administration of the cytotoxic agent cyclophosphamide to mice previously injected with a ga1E mutant of S. typhimurium, non-pathogenic to untreated animals, precipitated fatal infections due to multiplication of the ga1E strain. (ii) A "rough" mutant of S. dublin is in routine use in Great Britain as a live vaccine, given by parenteral injection, for protection of newborn calves against the frequently fatal Salmonella infections which were formerly prevalent; as the strain used appears to lack the 0-specific part of lipopolysaccharide, it presumably acts by invoking "non-specific immunity," perhaps by causing activation of macrophages.
Since live vaccines have substantially greater probability of success in providing for protection for the host against a subsequent invasion of a virulent wild strain, it is desirable to develop new live vaccines which avoid the shortcomings of vaccines prepared previously. Because the immune response of the vertebrate host to antigens, in particular surface antigens, of the pathogenic microorganism is the basic mechanism of protection by vaccination a live vaccine should retain the antigenic complement of the wild-type strain. The live vaccine should be non-virulent, substantially incapable of multiplication in the host, and should have substantially no probability for reverting to a virulent wild strain.
A non-virulent live vaccine may also serve as a host for the expression of antigens which may be located in the cytoplasm, translocated to the plasma or outer membrane or secreted to provide immunogens for an immunogenic response by the mammalian host. By employing a live vaccine as a carrier for an immunogen, particularly an invasive host, such as Salmonella typhi, a strong stimulus can be provided to the immune system, particularly to the humoral immune system. In this way many of the benefits of employing attenuated live pathogens, such as bacteria, fungi, protozoa and viruses can be achieved without concern for reversion to a virulent form.
2. Brief Description of the Prior Art
Sandhu et al., Infection and Immunity (1976) 13, 527 describes loss of virulence of Asperigillus fumigatus in a mutant auxotroph for p-aminobenzoic acid. Morris et al. Brit. J. Exptl. Path. (1976) 57:354 describes the effect of T and B lymphocyte depletion on the protection of mice vaccinated with a ga1E mutant of Salmonella typhimurium. Lyman et al., Inf. Imm. (1977) 15:491 compared the virulence of O:9,12 and O:4,5,12 S. typhimurium his.sup.+ transductants for mice. Descriptions of use of translocatable elements for causing deletions or inversions may be found in Kleckner, et al., J. Mol. Biol. (1977) 116, 125; Kleckner et al., ibid 127, 89; and Kleckner et al., Genetics, 90:427-464 (1978). U.S. Pat. No. 4,337,314 to Oeschger et al. describes the preparation of live H. influenzae vaccine strains by combining random mutations in a single strain. Hoiseth and Stocker, J. Bacteriol. (1985) 163:355-361, describe the relationship of aroA and serC genes of S. typhimurium.
In a private communication, Dr. John Roth, Department of Biology, University of Utah, developed two strains of S. typhimurium LT2, in each of which the transposon Tn10, conferring resistance to tetracycline, had been inserted into a gene of the aromatic biosynthetic pathway, thereby causing inability to synthesize the common precursor of the aromatic amino acids and of two bacterial metabolites, p-aminobenzoate, (precursor of the essential metabolite folic acid) and dihydroxybenzoate (precursor of the iron-chelating compound enterochelin or enterobactin). R. J. Yancey (1979) Infection and Immunity, 24, 174 report that a mutation causing inability to synthesize enterochelin secured in a mouse-virulent strain of S. typhimurium caused a very considerable reduction in virulence. The metabolic block was between chorismic acid and enterobactin, so that the mutation did not cause the requirement for p-aminobenzoate. In May, 1979, a paper was presented by Stocker and Hoiseth, entitled Effect of Genetic Defects in Iron Assimilation on Aromatic Biosynthesis on Virulence of Salmonella typhimurium.